RESPONSE OF in vitro CULTIVATED EGGPLANT (Solanum melongena L.) TO SALT AND DROUGHT STRESS

Transcription

1 AgroLife Sientifi Journal - Volume 6, Number 1, 217 ISSN ; ISSN CD-ROM ; ISSN ONLINE ; ISSN-L RESPONSE OF in vitro CULTIVATED EGGPLANT (Solanum melongena L.) TO SALT AND DROUGHT STRESS Ely ZAYOVA, Philip PHILIPOV, Trendafil NEDEV, Daniela STOEVA Institute of Plant Physiology and Genetis, 21, Aad. G. Bonhev Street, 1113 Sofia, Bulgaria Abstrat Corresponding author e_l_y@abv.bg Salinity and drought are some of the major abioti stresses that adversely affet eggplant produtivity and quality. The response of in vitro ultivated plants of three eggplant ultivars - Lagra Negra (LN), China-A2 (CH) and Blak Beauty (BB) to sodium hloride (NaCl) and polyethylene glyol (PEG 8 ) indued stress was studied. The Murashige and Skoog, 1962 nutrient medium (MS + 1% surose and.8% agar) supplemented with NaCl (; 5; 1; 15 and 2 mm) or PEG (; 3; 8 and 1%) was used. The survival rate, perentage of rooted plants, plant height and root length were reorded after three weeks of ultivation. The addition of 5 mm NaCl to the MS ulture medium influened positively the plants growth, development and rooting. The survival perentage signifiantly dereased while inreasing salt onentration in the ulture medium, espeialy at 1 mm NaCl. Conentration of 15 mm NaCl was toxi for the plants of all studied ultivars. The in vitro response of eggplant to drought stress was tested as well. The perentage of surviving plants also dereased while inreasing PEG onentration in the ulture medium. The appropriate onentrations of the abioti agent (1 mm NaCl for salinity and 8% PEG for drought) for in vitro seletion of tolerant eggplant plants were established. Salinity and drought due to NaCl and PEG respetively resulted in higher and more pronouned levels of proline in the ultivar LN plants, probably due to their better adaptation to abioti stress. Key words: eggplant, in vitro seletion, NaCl and PEG indued stress, survival rate, proline. INTRODUCTION Eggplant (Solanum melongena L.) is a vegetable rop speies belonging to the Solanaeae family. Eggplant possesses insuffiient levels of resistane to bioti (diseases and pests) and abioti (high salinity and drought) stress that ause serious rop losses. This problem has been addressed by hybridizing S. melongena L. with wild resistant Solanum speies, whih present a wide geneti diversity and are a soure of useful agronomi traits (Anisuzzaman et al., 1993; Blestos et al., 1998). However, this approah is limited by sexual inompatibility (Collonier et al., 21a) and diffiulties in obtaining fertile progenies (Gleddie et al., 1986). The appliation of the biotehnologial aspets of egg plant improvement has been used as a powerful tool for solving many target tasks in vegetable breeding. In vitro methods are suessfully applied to improve both the resistane to diseases and pests and the tolerane to abioti stress of eggplant (Collonier et al. 21b; Kashyap et al., 22; Kantharajah and Golegaonkar, 24). Eggplant tissues present high morphogeneti potential that makes this plant a suitable model for studies in different areas of plant siene (Magioli and Mansur, 25; Lakshman and Ravali, 216). Eggplant is moderately sensitive to salinity. The inreasing of NaCl negatively affets almost all aspets of plant development, inluding germination and vegetative growth (Akini et al., 24; Krusteva et al., 27). It affets as well the early growth harateristis of this speies. Growth parameters in seedlings and young plants are also influened by inreasing the NaCl (Akini et al., 24). The plant response to inreased salinity varies among speies and ultivars. Drought is one of the most important abioti stresses reduing rop growth and yield of eggplant. Tissue and ell ultures may be used to obtain plants tolerant to drought (Mohamed et al., 2). Polyethylene glyol (PEG), surose, mannitol or sorbitol are osmoti stress agents used in the in vitro seletion (Hassan et al., 24). However, the effet of proline aumulation in osmoti adjustment is still ontroversial and 276

2 varies aording to the type (Silveira et al., 23; Meloni et al., 24). Proline aumulation is a part of the adaptation to unfavorable environmental onditions, and a response to disturbane of the water balane of the ell at salt stress, low temperatures, heavy metals, and high aidity. Proline is synthesized in large quantities only when growth is sharply redued due to stress. The key enzyme responsible for the aumulation of proline in the ell is pyrroline-5-arboxylate synthetase; at stress onditions, this enzyme is aumulated in large quantities (Kavi Kishor et al., 1995). The aumulation of proline an be used as a biohemial marker for salt tolerane in eggplant. The aim of the present study was to investigate the influene of salinity (with NaCl) and drought (with PEG) on the plant growth, development and survival of in vitro ultured plants of different eggplant ultivars, and to selet forms with enhaned tolerane to the studied abioti fators. MATERIALS AND METHODS Initial plant materials and ulture medium The three eggplant ommerial ultivars, Larga Negra (LN); China-A2 (CH) and Blak Beauty (BB) used in the experiment were obtained from Institute of Plant and Geneti Resoures, Agriultural Aademy, Sadovo, Bulgaria. To study the effet of salinity and drought of the medium on the plant growth and development of the three eggplant ultivars, the basi Murashige and Skoog, 1962 medium (MS) ontaining 1% surose and.8% agar was used. This medium served as a ontrol. To indue salinity and drought, the ontrol medium was supplemented with ; 5; 1; 15 and 2 mm NaCl and PEG at onentrations of ; 3; 8, and 1%, respetively, with 4 plants. After appliation of the salt and drought stress to the three ultivars, the number of survived and rooted plants was ounted after 21 days of ultivation. The survival rate was defined as the ratio of the number of survived plants in eah ount and the number of the initially transferred plants. Morphologial parameters, suh as the height of the aerial plant part and the root length, were measured. The plants showed normal morphologial development, inluding minimal depression and development of the vegetation tip, whih was estimated by the normal growth of true leaves, at least.5 m in length. After this period of ultivation, the roots and the damaged leaves were removed and the plants were transferred to the same medium free of NaCl or PEG. Culture onditions All media were adjusted to ph 5.8 with 1N NaOH or HCl, prior to gelling with agar-agar and were sterilized by autolaving (121 C for 2 min.). Cultures were maintained in growth room at 24±1 C temperature, under 16/8 h light/dark regime, 4 μmol m 2 s 1 light intensity provided by white fluoresent light tube 36 W (Phillips). Content of free proline The ontent of free proline was determined aording to the method of Bates et al., 1973 in the ontrol and experimental variants on day 21 after indution of stress onditions. The extintion of the top layer, olored in pink, was measured spetrophotometrially at a wave length of 514 nm. The amount of proline was alulated by a standard urve (γ proline/ ml), and the resulting value was defined by the formula: E. V (ml toluene) [ mol/gfw] = FW (g) Statistial analysis The data were subjeted to one-way ANOVA analysis of variane for omparison of means, and signifiant differenes were alulated aording to Fisher s least signifiane differene (LSD) test at the 5% signifiane level using a statistial software pakage (Statgraphis Plus, version 5.1 for Windows). Data were presented as means ± standard error. RESULTS AND DISCUSSIONS The effet of NaCl stress on the three eggplant ultivars The effet of salt and drought stress on the growth of in vitro ultures, derived from seeds of three eggplant ultivars (LN, CH and BB) was examined. To assess the salt stress of the medium, the in vitro plants were transferred to a MS rooting medium ontaining, 5, 1, 15 and 2 mm NaCl for 21 days of ultivation. The plant growth was signifiantly 277

3 The onentration of 2 mm NaCl was toxi to the plants of the three studied ultivars. They were not able to overome the salt stress and died - a fat that indiates that they are very sensitive to high onentration of NaCl. Inhibition of the development of the vegetative tip and sorhing of leaves were also observed. Plant survival is often hosen as the main riterion for salt tolerane in eggplant (Akini et al., 24). This study has shown that the survival of in vitro ultivated plants depends on the ultivar and the stress intensity. Thus, the maintained a survival rate of 5% with 1 mm NaCl and was lassified as salt tolerant, and v. BB - as salt sensitive. Depending on the ultivars, high NaCl onentration affeted signifiantly the root growth. The positive effet of 5 mm NaCl onentration was demonstrated by the inreased plant height and root length of the three ultivars, whose dimensions were larger ompared to the ontrol plants (Table 2). Well-developed plants with dark green leaves were observed; the height of the aerial plant part was 4.5; 3.7 and 4.1 m for the three ultivars, and the roots length - 4.3, 4. and 3.7 m, respetively. At 1 mm NaCl onentration the plant height was signifiantly redued (2., 1.6 and 1.8 m, respetively for the three origins) and the root length dereased to 1.1; 1. and 1.5 m, due to nerosis observed in some of them. It was found that the root growth is more adversely affeted by the appliation of salt stress than the aerial plant part. redued with the inrease of NaCl onentration. The data about the influene of NaCl on survival rate, rooting and some morphologial parameters of the in vitro ultivated plants of the three eggplant ultivars are presented of Tables 1 and 2. In our studies, we searhed for this seletive onentration of NaCl, whih killed 7-8% of the plants, and allowed 2-3% of them to be viable for further development. The results show that the onentration of 5 mm NaCl in the ulture medium has a positive influene on the growth, development and rooting of the plants from all three tested origins. After 21 days of ultivation on ulture medium with 5 mm NaCl, 1% survival rate of all origins was registered. The plants were haraterized by well-developed leaves and roots. Upon addition of 1% NaCl in the ulture medium, the number of survived plants redued by almost half, sine part of the leaves turned yellow during the first week of ulture (Figure 1). During ultivation of the plants in the ulture medium with 15 mm NaCl, the plant number that survived salt stress signifiantly dereased. Only for 15% survival was reported; however, the leaves turned yellow and rooting was not observed after three weeks of ultivation (Figure 1). The few survived plants were haraterized with a very slow growth and by the end of the ulture period, the plant growth was terminated. These results showed that at this stage of in vitro development the was more tolerant to salinity ompared to the v. CH and v. BB. Table 1. Influene of NaCl on the survival of in vitro ultivated S. melongena plants Content of NaCl in the ulture medium Survived plants (%) v. CH Rooted plants (%) v. BB v. CH v. BB Control mm NaCl mm NaCl mm NaCl n = 4 plants 15 In fat, the slower growth is adaptive funtion for the plants survival under stress and the degree of salinity tolerane is often inversely proportional to the growth rate (Queiros et al., 27). The redution in growth not only helps the plant to onserve energy for tolerane but also limits the risk of inherited damage (Hossain et al., 27). During the further ultivation of the plants in the ulture medium free of NaCl, the harateristi morphologial type was reovered. The lak/presene of reparation apaity after stress may be used as one of the riteria for abioti stress tolerane (Yasar et al., 26). 278

4 Table 2. Influene of NaCl on the plant height and root length of in vitro ultivated S. melongena plants Plant height (сm) Root length (сm) Content of NaCl in the ulture medium Control 3.8± ±.86b 3.4±.89b 3.2±.81b 2.5±.71b 2.6±.78b d v. CH 5 mm NaCl 4.5± mm NaCl 2.±.65b 15 mm NaCl a 1.4±.57 v. BB v. CH v. BB 3.7± ± ± ± ± ±.28a 1.8±.46a 1.1±.38a 1.±.29a 1.5±.52a Data are presented as means of 4 plants per treatment ± standard error. Different letters indiate signifiant differenes assessed by the Fisher LSD test (5%) after performing ANOVA multifator analysis. Figure 1. Effet of NaCl on the growth and rooting of in vitro ultivated plants of S. melongena, There was a progressive aumulation of proline while inreasing the amount of NaCl (5, 1 and 15 mm) in the ulture medium ompared to a ontrol, in whih the levels of proline were signifiantly lower (Figure 2). At 15 mm NaCl this amino aid peaked. At the studied onentrations of NaCl, the lowest value of the biohemial markers was reported for the plants in v. BB, and the highest one in. Analysis of the plants indiated that the level of free proline has inreased in response to the inreasing onentrations of NaCl in the medium. A large inrease in response to salt stress (1 and 15 mm) was observed, although the growth of the plants at these onentrations was minimal. It has been found that the level of proline in the plants tolerant to saline stress is higher than that in the sensitive ones (Ashraf and Harris, 24). μmol/gf Larga Negra 2 China-A Control mm Figure 2. Content of proline in S. melongena, treated to salt stress The effet of PEG stress on the three eggplant ultivars Table 3 present details about the effet of PEG on the studied parameters in in vitro eggplant ultures. When the nutrient medium was supplemented with 3% PEG, the perentage of survived plants of all origins dereased. However, a signifiant differene with the 279

5 ontrol had not been proven. The perentage of rooting was almost equal to that in the ontrol medium for the three ultivars, i.e. this onentration has a positive impat on root growth and plants rooting (Table 3, Figure 3). Conentration of 8% PEG proved to be suitable for seleting plants that survived the drought stress of the medium. Survived plants were 6, 45 and 4% for the three ultivars respetively, and the rooted ones were 5, 4 and 3%. The treatment of the plants with 8% PEG for a period of 21 days has a strong reduing effet on the plants height and roots length. The plants with withered leaves, with signifiantly redued dimensions of the aerial plant part (1.9, 1.6 and 2. m, respetively for the three ultivars) and redued roots (1.5, 1.3 and 1.5 m, respetively) were observed on this nutrient medium (Table 4). The slower growth and development of the plants under drought onditions of the medium may be regarded as genetially onditioned adaptive response for their survival, while passing the stress effets (Yasar et al., 26). Using various onentrations of PEG, different behaviors depending on the ultivar were observed. The results showed that 8% PEG signifiantly slowed down the growth and dereased the plants height and the roots length of v. BB, while these morphologial parameters of were less affeted by this onentration. The depressive effet of PEG takes plae during root growth along the vegetative stage of development. The poor development of these parts is due to the inrease of the osmoti pressure in the medium and to nutritional imbalane of the plant aused by a lak of absorption and/or transport of nutrients to the stem (Evelin et al., 29). Table 3. Influene of PEG on the survival of in vitro ultivated S. melongena plants Content of PEG in the Survived plants (%) Rooted plants (%) ulture medium v. CH v. BB v. CH v. BB n = 4 plants Control % PEG % PEG % PEG 1 Table 4. Influene of PEG on the plant height and root length of in vitro ultivated S. melongena plants Content of PEG Plant height (сm) Root length (сm) in the ulture medium v. CH v. BB v. CH v. BB Control 4.2± ± ±.89 b 3.2± ±.85 b 2.6±.86 b 3% PEG 2.5±.83 b 2.2±.74 b 2.±.67 a 2.4±.74 b 2.±.61 b 2.1±.71 b 8% PEG 1.9±.51 b 1.6±.48 a 2.±.58 a 1.5±.44 a 1.3±.37 a 1.5±.47 a 1% PEG.8±.23 a Data are presented as means of 4 plants per treatment ± standard error. Different letters indiate signifiant differenes assessed by the Fisher LSD test (5%) after performing ANOVA multifator analysis. Figure 3. In vitro rooted plants of S. melongena, on MS medium а) ontrol and b) with 3% PEG 28

6 The impat of drought on free proline ontent was diretly dependent on the onentration of PEG as the rise in its level inreased the ontent of proline ompared to the ontrol. The differenes in the response of the three ultivars of eggplant to PEG indued stress was notieable, sine aumulated more proline than the other ultivars. From Figure 4 it is seen that the inreased onentration of the ating agent in the ulture medium led to an inrease of proline aumulation in the plants ompared to the ontrols, in whih the levels of proline were signifiantly lower. While higher levels of proline were estimated in plants, the lowest levels of proline were found in v. BB. μmol/gf Control 3% PEG 8% PEG Larga Negra China-A2 Figure 4. Content of proline in S. melongena, treated with PEG The experimental results were similar for both types of abioti stress - salinity and drought, as observed by the varietal differenes. The higher ontent of proline in showed some plastiity in its response to abioti stress appliations. The results obtained are in aordane with those reported by several other authors: the drought onditions inhibit the growth proesses of the plants, leading to a number of metaboli hanges. CONCLUSIONS The seletive onentrations of 1 mm NaCl for salinity and 8% PEG for drought of the medium were identified. These in vitro stress onditions allow rapid sreening of eggplant ultivars with inreased tolerane to salinity and drought. Cv. LN overomes in a greater extent both stresses of the ulture medium in omparison with the other two origins v. CH and v. BB. The presented in vitro model an be used for preliminary evaluation of different eggplant genotypes tolerane to salinity and drought of the ulture medium. The aumulation of proline is onsidered a marker for abioti stress, proteting tissues from damage. REFERENCES Akini I., Akini S., Yilmaz K., Dikii H., 24. Response of eggplant varieties (Solanum melongena L.) to salinity in germination and seedling stages. New Zealand Journal of Crop and Hortiultural Siene, 32, p Anisuzzaman M., Kamal H.M., Islam R., Hossain M., Joarder O.I., Genotypi differenes in somati embryogenesis from hypootyls explants in Solanum melongena L., Plant Tissue Culture, 3, p Ashraf M., Harris J.C., 24. Potential biohemial indiators of salinity tolerane in plants. Plant Siene, 166, p Bates L.S., Waldren R.B., Teare E.D., Rapid determination of free proline for water stress studies. Plant and Soil, 39, p Blestsos F.A., Roupakias D.G., Tsaktsira M.L., Saltsojannes A.B., Thanassoulopoulos C.C., Interspeifi hybrids between three eggplant (Solanum melongena L.) ultivars and two wild speies (Solanum torvum Sw. and Solanum sisymbriifolium Lam.). Plant Breeding, 117, p Collonnier C., Mulya K., Fok I., Mariska I., Servaes A., Vedel F., Souvannavong V., Dureux G., Sihahakr D., 21a. Soure of resistane against Ralstonia solanaeraum in fertile somati hybrids of eggplant (Solanum melongena L.) with Solanum aethiopium L., Plant Siene, 16, p Collonnier C., Fok I., Kashyap V., Rotino G.L., Daunay M.C., Lian Y., Mariska I.K., Rajam M.V., Servaes A., Dureux G., Sihahakr D., 21b. Appliations of biotehnology in eggplant, Plant Cell, Tissue and Organ Culture, 65, p

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